Toll like receptors (TLRs) on eukaryotic cells mediate the recognition of microbial molecules and regulate host responses to microbial pathogens. Proper regulation of TLR signals not only restricts the intensity and duration of inflammatory responses, but may also maintain immune homeostasis in the intestine, where a plethora of microbial pathogens co-exist with host cells. Recent studies from our labs indicate that A20 is a novel protein that is required for terminating TLR induced signals on myeloid cells and for preventing excessive inflammatory responses in vivo. Our preliminary data suggest that A20 is essential for restricting TLR signals in vivo. Moreover, our findings suggest that A20 may be a novel enzyme that performs this critical function by directly modulating the ubiquitylation status of TLR signaling proteins such as TRAF6. These modifications may cause both de-activation and degradation of TLR signaling proteins. These exciting preliminary findings provide us with unique opportunities to test our central hypothesis that A20 regulates ubiquitylation of signaling proteins, TLR signals, and immune homeostasis. This application represents a synergistic effort between the genetic and cellular expertises of the Ma lab and the biochemical expertise of the Pickart lab to determine: (i) whether A20 is essential for regulating TLR responses in mice;(ii) whether A20 regulates ubiquitylation of critical TLR signaling proteins;and (iii) how A20 recognizes ubiquitylated proteins and may function as both a de-ubiquitylating enzyme and an E3. Results from these studies promise to yield significant insights into how A20 links the regulation of ubiquitylation with disease.